6-(alpha-(guanylureidoalkanoylamino)aracylamino)penicillanic acids

ABSTRACT

2-(HOOC-),3,3-DI(H3C-),6-(R2-N(-R3)-C(=N-R4)-N(-R1)-CO-   (NH-A-CO)N-NH-CH(-AR)-CO-NH-)-PENAM COMPOUNDS OF THE FOROULA WHEREIN AR IS PHENYL, 4-HYDROXYPHENYL, 2-THIENYL OR 3THIENYL, A IS ALKENE OF 1 TO 4 CARBON ATOMS OR ALKYLIDENE OF 2 TO 4 CARBON ATOMS, N IS AN INTEGER OF 1 TO 3, R1, R2, R3 AND R4 CONSIDERED SEPARATELY ARE EACH HYDROGEN OR ALKYK OF 1 TO 4 CARBON ATOMS, R1 AND R4 CONSIDERED TOGETHER ARE ALKYLENE OF 2 TO 4 CARBON ATOMS, R2 AND R4 CONSIDERED TOGETHER ARE ALKYLENE OF 2 TO 4 CARBON ATOMS; AND R2 AND R3 CONSIDERED TOGETHER ARE ALKYLENE OF 4 TO 5 CARBON ATOMS AS BROAD SPECTRUM ANTIBIOTICS.

"United States Patent Oihce 3,838,153 6-[a-(GUANYLUREIDOALKANOYLANIINO)ARACYLAMINOFENICILLANIC ACIDS Ernest S. Hamanaka, Groton, and David S.Patton, North Stonington, Conn., assignors to Pfizer Inc., New York,

Nb iirawin Filed May 16, 1972, Ser. No. 253,856 rm. (:1. (107d 99/16 US.Cl. 260-2391 11 Claims ABSTRACT OF THE DISCLOSURE Compounds of theformula Ar-OHC ONH S on, \/CH3 wherein Ar is phenyl, 4-hydroxyphenyl,2-thienyl or 3- thienyl; A is alkylene of 1 to 4 carbon atoms oralkylidene of 2 to 4 carbon atoms; n is an integer of 1 to 3; R R R andR, considered separately are each hydrogen or alkyl of 1 to 4 carbonatoms, R, and R considered together are alkylene of 2 to 4 carbon atoms;R and R considered together are alkylene of 2 to 4 carbon atoms; and Rand R considered together are alkylene of 4 to 5 carbon atoms as broadspectrum antibiotics.

BACKGROUND OF THE INVENTION variable and possess the general formulaindicated below on, s 2 n--d:--Nrr 5/ cm -co H 7 a Z o 4 wherein theacyl moiety on the 6-aminopenicillanic acid is derived from a carboxylicacid or functional derivative thereof such as an acyl halide oranhydride.

The pharmacodynamic properties and antibiotic profile of a givenpenicillin are determined to a large extent by the nature of the Rgroup. The most widely used penicillins are those wherein the R moietyis represented by benzyl-, phenoxymethyland a-phenoxyethyL. While thesewell-known analogs are highly antagonistic toward grampositivemicro-organisms they have limited gram-negative activity. Consequently,drugs which will combat rise in gram-negative infections, e.g., E. coli,Pseudomonas or Klebsiella, are of value to the medical profession.

Recent efforts to improve the profile of activity within the family ofpenicillins has resulted in the synthesis of several new agents.u-Carboxybenzylpenicillins (U.S. Pat. 3,142,673), a broad spectrumantibiotic, is reported to have greater efficacy against gram-negativebacteria via the parenteral route of administration, but has limitedutility via oral administration. a-Aminoarylmethylpenicil- 3,838,153Patented Sept. 24, 1974 lins and congeners thereof (U.S. Pats.2,985,648, 3,140,- 282, 3,373,156, 3,308,023 and 3,342,677) are known,but have a limited spectrum of activity against certain gramnegativemicro-organisms. Both gram-negative and grampositive activity areclaimed for 6-ureidopenicillanic acid derivatives in US. Pats.3,180,863, 3,120,512 and 3,118,- 877 and for a-urediopenicillins in US.Pat. 3,352,851. Activity against gram-negative bacteria, especiallythose of the genus Pseudomonas, is claimed fora-carbamylureidopenicillins (U.S. Pat. 3,483,118) anda-alkoxycarbonylureidopenicillins (U.S. Pat. 3,481,922). More recently,a-guanylureidopenicillins have been reported to be useful againstinfectious diseases, especially those caused by the Pseudomonas genus(U.S. Pat. 3,579,501 and Belgium Specification 742,423).

SUMMARY OF THE INVENTION and the pharmaceutically acceptable basic saltsthereof, wherein Ar is phenyl, 4-hydroxyphenyl, 2-thienyl or 3- thienyl;n is an integer of 1 to 3; A is alkylene containing from 1 to 4 carbonatoms or alkylidene containing from 2 to 4 carbon atoms; R R R and Rwhen considered separately are each hydrogen or alkyl containing from 1to 4 carbon atoms; R, and R when considered together are alkylenecontaining from 2 to 4 carbon atoms; R and R when considered togetherare alkylene containing from 2 to 4 carbon atoms; and R and R whenconsidered together are alkylene containing from 4 to 5 carbon atoms,are potent antibacterial agents, especially against gramnegativemicro-organisms.

A preferred group of congeners are those wherein n is 1, A is alkyleneor alkylidene of the above-mentioned carbon limitations, R R R and R areeach hydrogen or alkyl and Ar is phenyl or Z-thienyl.

A second preferred class of compounds of the instant invention are thosewherein Ar is phenyl, n is 2 or 3 and A is alkylene of the aforesaidcarbon limitation.

As one skilled in the art can readily appreciate, the u-carbon atom ofthe penicillin side chain to which the guanylureidoalkanoylamino moietyis attached is an asymmetric carbon atom allowing for the existence oftwo optically active isomers, the D- and L-diasteroisomers, as well asthe racemate, DL form. In accord with previous finding concerning theactivity of such penicillins possessing asymmetric u-carbon atoms, thecompounds of the present invention possessing the D-configuration aremore active than those of the L-configuration and are the preferredcompounds, although the L and DL forms of the instant compounds are alsoconsidered within the purview of the present invention.

Compounds of the instant application wherein A is alkylidene, derivedfrom an u-amino acid, have an asymmetric carbon atom allowing for D, Land DL forms. Although the natural form of the starting amino acid, theL form, is the preferred, the DL and D are considered with in the scopeof the present invention.

It is noteworthy to mention while considering asymmetric centers, thatthere are several in the 6-aminopenicillanic nucleus, the basic buildingblock from which the compounds of the instant invention are derived.These potential additional isomers are not significant in this instancesince the 6-aminopenicillanic acid employed as the starting material isthat which is produced by fermentation and is consistently of oneconfiguration.

Also considered within the purview of the present invention are3-(1-alkanoyloxyalkyl) esters of the subject compounds as well ascompounds of the aforementioned formula wherein, in addition to thementioned variables, Ar is 1,4-cyc-lohexadien-l-yl or can be cycloalkylof -7 carbon atoms; A is cycloalkylene, cycloalkylidene, arylene,aralkylene, alkylenearalkylene, alkylene or alkylidene substituted byhydroxy, mercapto or methythio or alkylenearylene; A taken together withthe nitrogen of the urea moiety forms a heterocyclic ring; and congenerswherein R R R and R are each phenyl or benzyl, or phenyl and benzylsubstituted by one or more organic radicals.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the processemployed for synthesizing the penicillins of the present invention twopreparative routes are amenable. The first is illustrated as follows:

In practice, the requisite a-aminoarylmethylpenicillin and acidchloride, wherein Ar, A, R R R and R are previously indicated and n=l,are contacted in a reaction-inert, aprotic solvent in the presence of ahydrogen halide scavenger, such as a tertiary amine, at ice-bathtemperatures. Equimolar amounts of the reactants are generally employedwith as much as a -100% excess of the acid chloride. Generally, when onemole each of the reactants are employed, three moles of a tertiaryamine, preferably triethylamine, are used. If additional acid chlorideis added, a corresponding molar amount of amine is employed.

The reaction-inert solvent comprising the liquid phase of said reactionmixture should be one which does not react to any appreciable extentwith either the reactants or product of said reaction. The preferredsolvents should be anhydrous, aprotic, polar solvents such asdimethylformamide or hexamethylphosphoramide.

Although the initial contacting of the reactant is carried out atice-bath temperature, in order to reduce the incidence of by-products,it is desirable after a few minutes of mixing to allow the reactionmixture to warm to room temperature until the reaction is complete ornearly complete. The reaction time, which will vary depending ontemperature, concentration and inherent reactivity of the startingreagents, is usually from 0.5 to 4 hours.

On completion of the above-mentioned reaction, any insolubles arefiltered and the product is precipitated by adding the filtrate to alarge volume of diethyl ether. The

crude product is isolated by suction filtration and drying. A suspensionof the crude product in methylene chloride is rendered free of any traceamounts of starting tat-aminoarylrnethylpenicillin by the addition of asmall amount of triethylamine, which converts the starting penicillin t0the methylene chloride soluble triethylamine salt. The pure product issubsequently filtered and dried.

The starting reagents leading to the products of the present inventionare easily prepared by methods familiar to those skilled in the art. Thea-aminoarylmethylpenicillins are known and described in US. Pats.2,985,- 648 and 3,342,677 and by Long, et al., J. Chem. Soc., 1920(1971), while the 3-guanyl-l-ureidoalkanoic acids are synthesized eitherby the procedure of Frankel, et al., J. Chem. Soc., 2698 (1967) orShapiro, et al., J. Am. Chem. Soc., 81, 2220 (1959).

The alternate synthetic procedure employed in the preparation of thecompounds of the present invention wherein Ar, A, n, R R R and R are aspreviously indicated, is illustrated as follows:

In accordance with the above outlined synthetic scheme, equimolaramounts of the requisite 3-guanyl-lureidoalkanoic acid,N-hydroxysuccinimide and dicyclohexylcarbodiimide DCC), plus as much asa 50% excess of the two latter reagents, are contacted in areactioninert solvent, such as those indicated in the previouslydiscussed preparative procedure, at ambient temperatures. After 1 to 3hrs, the intermediate N-succinimido ester generated in sitn, is treatedwith an equimolar amount, plus as much as a 50% excess, of theappropriate oraminoarylmethylpenicillin, preferably as a basic salt. Thereaction time can vary from 1 to 6 hrs. depending on the temperature,concentration and reactivity of the reagents employed.

The product is isolated, after filtration of the reaction mixture, byaddition of the reaction filtrate to a large volume of diethyl ether.The product can be purified,

when required, by employing triethylamine as mentioned in the previouslydiscussed preparative method.

The starting materials employed in this second synthetic procedureleading to the compounds of the present invention are either commercialreagents or are prepared by methods familiar to those skilled in theart.

As one skilled in the art can recognize, the guanidine moiety of theinstantly claimed compounds can exist in several diiferent tautomericforms, all of which are considered within the purview of the presentinvention.

As has been previously noted, a characteristic feature of the acidiccompounds of the instant invention is their ability to form basic salts.Acid congeners of the present invention are converted to basic salts bythe interaction of said acid with an appropriate base in an aqueous ornonaqueous medium. Such basic reagents suitably employed in thepreparation of said salts can vary in nature, and are meant tocontemplate such bases as organic amines, ammonia, alkali metalhydroxides, carbonates, bicarbonates, hydrides and alkoxides, as well asalkali earth metal hydroxides, hydrides, alkoxides and carbonates.Representative of such bases are ammonia, primary amines such asn-propylamine, n-butylamine, aniline, cyclohexylamine, benzylamine,p-toluidine, ethylamine, octylamine, secondary amines such asdicyclohexylamine and tertiary amines such as diethylaniline,N-methylpyrrolidine, N-methylmorpholine and1,5-diazabicyclo-[4,3,01-5-nonene; sodium hydroxide, potassiumhydroxide, ammonium hydroxide, sodium ethoxide, potassium methoxide,magnesium hydroxide, calcium hydride and barium hydroxide.

As one skilled in the art can readily appreciate, the compounds of theinstant invention are sufficiently basic, by virtue of the guanylmoiety, to form acid addition salts; said salts are also consideredwithin the scope of this invention.

In the utilization of the chemotherapeutic activity of those compoundsof the present invention which form basic salts, it is preferred, ofcourse, to use pharmaceutically acceptable salts. Although waterinsolubility, high toxicity, or lack of crystalline nature may make somesalt species unsuitable or less desirable for use as such in a givenpharmaceutical application, the water insoluble or toxic salts can beconverted to the corresponding acids by decomposition of the salts asdescribed above, or alternately they can be converted to any desiredpharmaceutically acceptable basic salt. The said pharmaceuticallyacceptable salts preferred include the sodium, aluminum, potassium,calcium, magnesium, ammonium and substituted ammonium salts, e.g.,procaine, dibenzylamine, N,N- bis(dehydroabietyl)ethylenediamine, 1ephenamine,N- ethylpiperidine, N-benzyl-fl-phenethylamine,N,N'-dibenzylethylenediamine, triethylamine, as well as salts with otheramines which have been used to form salts with benzylpenicillin.

The novel penicillins described herein exhibit in vitro activity againsta wide variety of microorganisms, including both gram-positive andgram-negative bacteria. Their useful activity can readily bedemonstrated by in vitro tests against various organisms in abrain-heart infusion medium by the usual two-fold serial dilutiontechnique. The in vitro activity of the herein described compoundsrenders them useful for topical application in the form of ointments,creams and the like, or for sterilization purposes, e.g., sick-roomutensils.

These novel penicillins are also efliective antibacterial agents in vivoin animals, including man, not only via the parenteral route ofadministration but also by the oral route of administration.

Obviously, the physician will ultimately determine the dosage which willbe most suitable for a particular individual person, and it will varywith the age, weight and response of the particular patient as well aswith the nature and extent of the symptoms and the pharmacodynamiccharacteristics of the particular agent to be administered. It willoften be found that when the composition is administered orally, largerquantities of the active ingredient will be required to produce the samelevel as produced by a small quantity administered parenterally.

Having full regard for the foregoing factors it is considered that aneffective daily oral dose of the compounds of the present invention inhumans of approximately l0- mg./kg. per day, with a preferred range ofabout 50-75 mg./kg. per day in single or divided doses, and a parenteraldose of 25-100 mg./kg. per day, with a preferred range of about 20-75mg./kg. per day will effectively alleviate the symptoms of theinfection. These values are illustrative, and there may, of course, beindividual cases where higher or lower dose ranges are merited.

As has been previously mentioned, the penicillins of the presentinvention are broad spectrum antibiotics which are, unlike manypenicillin analogs, highly antagonistic toward gram-negativemicroorganisms, in particular LE. coli, 'Psew domonas and Klebsiella.Further, they appear to be more resistant than most penicillins todestruction by penicillinase, an enzyme produced by certain bacteriawhich degrades penicillin to an inactive penicillanic acid.

The preferred compounds of the present invention are 6-[2-phenyl-2-(3-guanyl 1 ureidoacetamido)acetamido] penlcillanic acid,6-[2-phenyl-2-(fi-ES-guanyl-Lureidopropionamido)acetamido1penicillanicacid, 6-[2-(2-thienyl)- 2-(3-guanyl 1ureidoacetamido)acetamido]penicillanic acid,6-[2-phenyl-2-(3-guanyl-1-ureidoacetylglycinamido)-acetamido1penicillanic acid, 6 [2 phenyl 2 (3-guanyl- 1ureidoacetylglycylglycinamido)acetamido]penicillanic acid and6-[2-phenyl-2-(3-{N-methylguanyl}-1-ureidoacetamido)acetamido]penicillanicacid. Further preferred within this group of outstandin compounds arethose diasterioisomers of the D-configuration.

The antimicrobial spectra of a number of compounds of the instantinvention are provided in the following tables. Table I compares the invitro profile of 6-[D-2- phenyl 2(3-guanyl-l-uredioacetamido)acetamido]penicillanic acid (PGU) withampicillin (AMP) and carbenicillin (CAR). The tests were run understandardized conditions in which nutrient broth containing variousconcentrations of the test material was seeded with the particularorganism specified, and the minimum concentration (MIC) at which growthof each organism failed to occur was observed and recorded.

TABLE I In vitro Comparison Data for PGU, AMP, and CAR (MIC; megJml.)

MIC

Organism PGU AMP CAR E. coli 511 .266 .3 E. coli smooz 20% 251 1 22i)?!Pa. aeruginosa 521x490. 0. 39 0. 78 0. 6 Ps. aeruginosa 521 .104. 3. 12200 50 P8. aeruginosa 52A173- 3. 12 38 75 Kleb. pneumoniae 53A(] 25 100200 Kleb pneumoniae 535.015 25 250 200 A. aerogenes 55A002 0. 78 100 25A. aerogenes 55A004--- 0.78 5 50 S21. marcescens 631 .001 1. 56 200 25.P. mzmbilis 57C015 0.78 1. 56 1. 25 P. vulgaris 57A059 0.78 6 12. 5 S.aureus 01A005 0. 19 0. 09 1. 56

Table 11 presents the in vivo comparison data for the three compoundscontained in Table I against several experimental infections in mice.

The values (percent of survivors) are obtained under standard conditionsknown to those skilled in the art. For the E. coli organism, the testcompounds is administered to the infected mice by a multiple dosingregimen in which the first dose is given 0.5 hour after inoculation andis repeated four and twenty-four hours later. Against Ps. aeruginosa,the test compound is administered at 1, 3, 5, 7 and 24 hrs. afterinoculation.

TABLE II Percent protection 3 Dose Organism Route 1 (mg/kg.) PGU AMP CARE. coli 51A266 PO 200 50 90 70 50 30 80 10 E. coli 51.4266 SC 200 90 9090 50 70 80 90 Ps. aeruainosa 62A104..... SC 400 90 80 1 PO=ral;SC=subcutaucous route of administration. I Percent of survivors.

Table III presents additional in vitro data for related compounds of theinstant invention.

TABLE III S Q-pnoonn on. NH I CH3 (J N.

: COaH Hz 11 NH MIC (meg/ml.)

Organism n=1 n=2 n=3 S. aureus 01A005 0. 19 0. 73 3.12 S. aureus0111006...- 0. 78 0. 78 1. 56 S. aureus 01A212 25 50 50 Strep. pyogenea020203 0. 01 0. 098 0. 012 Pa. aerugz'nosa 52Al04 3. 12 6. 25 25 Ps.aeruqinosa 52A490 0. 39 0. 78 3. 12 K leb. pneumoniae 53A009 25 100 200H. influenzae 54A001--. 0.09 0. 39 0. 39 A. aerogems 55A002. 0.78 35 50.P. mirabilis 570015 0.78 12. 50 Sal. cholerae-suis 58B242 1. 5 12. 5 25Ser. murcescens 63A001 1. 56 50 200 Table IV presents in vivo comparisondata for the three compounds in Table III against E. coli infections inmice.

The values, reported as percent survivors, are obtained under standardconditions familiar to those skilled in the art. The test compound isadministered to the infected mice by a multiple dosing regimen in whichthe first dose is given 0.5 hour after inoculation and is repeated fourand twenty-four hours later.

The novel products of this invention are of value as antibacterialagents and are remarkably effective in treating a number of infectionscaused by susceptible gramnegative and gram-positive bacteria in poultryand animals including man. For such purposes, the pure materials ormixtures thereof with other antibiotics can be employed. They may beadministered alone or in combination with a pharmaceutical carrier onthe basis of the chosen route of administration and standardpharmaceutical practice. For example, they may be administered orally inthe form of tablets containing such excipients as starch, milk, sugar,certain types of clay, etc., or in capsules alone or in admixture withthe same or equivalent excipients. They may also be administered orallyin the form of elixirs or oral suspensions which may contain flavoringor coloring agents, or be injected parenterally, that is,intramuscularly or subcutaneously. For parenteral administration, theyare best used in the form of a sterile aqueous solution which may beeither aqueous such as water, isotonic saline, isotonic dextrose,Ringers solution, or non-aqueous such as fatty oils of vegetable origin(cotton seed, peanut oil, corn, sesame) and other nonaqueous vehicleswhich will not interfere with the therapeutic efficiency of thepreparation and are nontoxic in the volume or proportion used (glycerol,propylene glycol, sorbitol). Additionally, compositions suitable forextemporaneous preparation of solutions prior to administration mayadvantageously be made. Such compositions may include liquid diluents,for example, propylene glycol, diethyl carbonate, glycerol, sorbitol,etc.; buffering agents, as well as local anesthetics and inorganic saltsto alIord desirable pharmacological properties.

The following examples are provided solely for the purpose ofillustration and are not to be construed as limitations of thisinvention, many variations of which are possible without departing fromthe spirit or scope thereof.

EXAMPLE 1 6-[D-2-Phenyl-2-(3-guanyl 1ureidoacetamido)acetamido]penicillanic Acid (Ar=; A=CH n=1; R R R and R=H) A. To a suspension of 12.7 g. (0.0325 mole) ofD-uaminobenzylpenicillin trihydrate suspended in 200 ml. ofdimethylformamide at room temperature and under a nitrogen atmosphere isadded 9.15 ml. of triethylamine, and the resulting solution cooled to 0C. in an ice bath. Over a period of 2 min. two 3.5 g. portions (0.0325mole) of guanylureidoacetyl chloride hydrochloride are added to thereaction mixture with vigorous stirring and the mixture stirred at 0 C.for 5 min. and at room temperature for 50 min. The mixture is cooledagain in an ice bath and 4.55 ml. of triethylamine and an additional two3.5 g. portions of the acid chloride added over a 2 min. period. Afterstirring at 0 C. for 10 min. and at room temperature for 1 hr., themixture is filtered and the filtrate added to 3 l. of diethyl ether. Thecrude product, 19.5 g., is suspended in 200 ml. of methylene chloridecontaining 5 ml. of triethylamine and allowed to stir for 2 hrs. Theproduct, a white powder, is filtered and dried, 14.0 g. (88% yield).

Infrared spectrum peaks (microns; KBr): 3.0, 3.5, 5.6, 5.75-6.3 (b),6.3-6.75 (b), 7.2, 7.6, and 8.1.

Nuclear magnetic resonance spectrum peaks (PPM; DMSO-D 1.5 (d), 6H; 3.9(c), 2H; 4.0 (s), 1H; 5.4 (c), 2H; 5.8 (c), 1H; 7.4 (c), 7H; and 8.4-9.2(c), 5H.

B. To a solution of 345 mg. (3 mmoles) of N-hydroxysuccinimide and 620mg. (3 mmoles) of dicyclohexylcarbodiimide in 15 ml. of drydimethylformamide is added 587 mg. (3 mmoles) of 3-guanyl-l-ureidoaceticacid hydrochloride, and the mixture allowed to stir at room temperaturefor 2 hrs. D-a-Aminobenzylpenicillin triethylamine salt (1.35 mg., 3mmoles) is added and stirring is continued for an additional 3 hrs.Dimethylformamide (20 ml.) is added to the reaction mixture followed byfiltration and addition of the clear filtrate to 1.2 l. of diethylether. The dried white precipitate, 947 mg., isolated by suctionfiltration, is suspended in 50 ml. of methylene chloride and treatedwith one ml. of triethylamine. After one hr. of stirring, the purifiedproduct is collected and dried in vacuo, 537 mg. (36.5% yield).

The product is identical in every respect with that isolated via theacid chloride preparation procedure of Example 1-A.

EXAMPLE 2 6-[D-2-Phenyl-2- (,8 3 guanyl 1 ureidopropionamido)acetamido]penicillanic Acid (Rr=; A=--(CH "=1; R1, R2 R3 andfi-(S-Guanyl l ureido) propionic acid hydrochloride (840 mg.; 4 mmoles)is added to a solution of 785 mg. (3.8 mmoles) ofdicyclohexylcarbodiimide and 440 mg. (3.8 mmoles) ofN-hydroxysuccinimide in 15 ml. of dry dimethylformamide at roomtemperature and under a nitrogen atmosphere, and the mixture allowed tostir for 10 solubles are filtered, and the filtrate poured into 1 l. ofdiethyl ether. The precipitated product, after stirring for 30 min., isfiltered and dried. It is further purified using methylene chloride andtriethylamine as in Example 1.

EXAMPLE 4 Starting with the appropriate u-aminoarylmethylpenicillin andguanylureidoalkanoyl chloride hydrochloride, and employing the generalprocedure of Example 1, the following penicillins of the configurationshown are synthesized:

o 3 CH: ArOHOONH- X I CH: NH L corn 1 In this and subsequent tables, the1 8 group representing alkylene is written in such a manner that theleft II bond of the group is attached to the -C group and the right bondof the group to the NH atom.

(s), 1H; 5.4 (c), 2H; 5.75 (c), 1H; 7.1-7.8 (c), 7H; and 8.3-9.3 (0),5H.

EXAMPLE 3 6 [D 2 "(2 Thienyl) 2 (guanylureidoacetamido)acetamido]penicillanic acid (Ar=2 thienyl; A=CH n=l; R R R and R =H) A150 m1. flask fitted with a nitrogen bleed is charged with 3.55 g. (0.01mole) of D-a-amino-2-thienylmethy1- penicillin suspended in 70 ml. ofdry dimethylformamide, and the resulting suspension treated with 3.06ml. (0.022 mole) of triethylamine. The reaction mixture is cooled to' 0C. and treated with 2.35 g. (0.011 mole) of guanylureidoacetyl chloridehydrochloride added portionwise over a period of 2 min. The suspensionis allowed to warm to room temperature and remain at this temperaturefor min., after which the mixture is treated with an additional 1.5 ml.(0.011 mole) of triethylamine, cooled to 0 C. and treated with 2.35 g.(0.011 mole) of the acid chloride portionwise. After stirring at 0 C.for 10 min., the mixture is allowed to warm to room temperature andremain at this temperature for 1-2 hrs. The small amount of in- EXAMPLE5 6-[-D-2-'Pheny1-2-(3-{N-methylguanyl}l-ureidoacetamido) A' suspensionof 2.25 g. (5 moles) of D-a-aminobenzylpenicillin triethylamine salt in25 ml. of dry dimethylformamide, maintained under a nitrogen atmosphere,is treated with 0.7 ml. (5 moles) of triethylamine, and the reactionmixture cooled to --30 C. 3-(N-methylguany1)-1-ureidoacetyl chloridehydrochloride (2.28 g.,

0.01 mole) is added in divided portions over a period 6 ['D2-Phenyl-2-(3-guanyl-l-ureidoacetylglycinamido)- acetamido[penicillanicacid (Ar=; A=CH 111:2; 1. 2 3 and R4=H) To 175 m1. of dimethylformamide,maintained under a nitrogen atmosphere, is added 4.3 g. (17 moles) of3-guanyl-l-ureidoacetylglycine hydrochloride followed by the addition of2.42 g. (21 mmoles) of N-hydroxysuccinimide and 4.35 g. (21 mmoles) ofN,N'-dicyclohexylcarbodiimide. The resulting suspension is allowed tostir for 2.5 hrs. and is then filtered. D-u-Aminobenzylpenicillintriethylamine salt (9.45 g., 21 mmoles) is added to the filtrate and themixture allowed to stir at room temperature for 2 hrs. The mixture isfiltered and the filtrate added dropwise over a 20 min. period to 2.5 l.of diethyl ether. adfled Six drops of triethylamine' Afler Stirring for30 The resulting precipitate is stirred for 20 min., filtered and theproduct i filtered and d in vacuo, 310 slurried in methylene chloride towhich is added 4.5 ml. Infrared spectrum peaks (microns; KBr): 3.05,5.65, of triethylamine. After 25 min. the product, as a tan 6'05 655 720and solid, is filtered and vacuum dried, 8.9 g. v Infrared SpectrumPeaks (microns; KBr): 105 5.65, Nuclear magnetic spectrum peaks (PPM,DMSO-D O). 6.04, 6.58, 7.20, 7.60 and 8.10, 1.40, (6H); 3.20-4.1 (b);3.38, (1H); 5.7, (1H); and

Nuclear magnetic resonance spectrum peaks (PPM: 7.34, D O): 1.32, 6H;3.85; 6.35, (2H); and 7.32, (5H). EXAMPLE 10 EXAMPLE 9 Starting with therequisite guanylureidoalkanoylaminm 5- 1)2-:Pheny1-2-=(B-guanyl-1.urgidgagetylglycylglycina. acid orguanylureidoalkanoyldipeptide and DL-a-amimido)-acetamido]penicillanicacid (Ar==; A=CH noarylmethylpenicillin triethylamine salt and following1, 2 3 and 4= the general procedure of Example 8 or 9, the following Toa solution of 310 mg. (1 mmole) of 3-guanylureidocompounds are prepared:

Ar-CHC ONH CH: l CH3 5 N C0zH NH 1 R, O=( JN--GN\ Ar A 1| R1 R4 R: R;

c H c efitti 40%.: i i E CqHs- -CH(CH )CHr- 2 H- H- H- CH3- CuHs- CHH 2H- H-- n-C H1- fl-C H1 c H I 5 CH:(BH 3 H 02H: H CzHs c H -CH CH C:H:C1t. 5 5.H. 115" 8%;

CGHISCHI-(EHCHF 4-HOCe P (C 2)z 2 H E Qa- 2):

4-HOCIHr- CHCHKI:H 2 H- z-oim- H- CH3- 4-HOC6Hr' CHCHAIHF 2 H-- H-C(HBCHP ctHioHr- 4-HootHt- '-cH2CH(CH1)- a H- i-CaHz- H- #03111-4-HOCQHL- 2 H- H- -(CH=)t- 6-[D-2-Phenyl 2(B-guanyl-l-ureidoacetamido)acetamido]penicillanic acid,3-pivaloyloxymethyl ester Triethylamine (1.4 ml., .01 mole) is added toa solution of 2.5 g. (.005 mole) of D-a-aminobenzylpenicillin-3-pivaloyloxymcthyl ester hydrochloride in 50 ml. of drydimethylformamide and the resulting solution, which is maintained undera nitrogen atmosphere, is cooled to C. in an ice bath and treated with1.07 g. (.005 mole) of 3-guanyl-l-ureidoacetyl chloride. After stirringthe reaction for min. at ice bath temperature, an additional 0.35 ml.(.0025 mole) of triethylamine and 535 mg. (.0025 mole) of the acetylchloride are added. This procedure is repeated again wherein 0.18 ml.(.0013 mole) of the amine and 278 mg. (.0013 mole) of the acid chlorideare added. After stirring for the final 30 min. the reaction mixture isfiltered and filtrate poured into the 1.1 1. of

S CH; Al'-CHC ONE- I CH in approximately 20 ml. of methylene chlorideand reprecipitated with 500 ml. of diethyl ether. Filtration and dryingprovides the desired product as a white powder, 2.05 g. (81.5% yield).

Infrared spectrum peaks (microns; KBr): 3.0, 3.4, 5.6 (sh), 5.65, 5.75,5.85-6.3(b), 6.3-6.8(b), 6.85, 7.15, 7.3, 7.7(sh), 7.8, 8.1, 8.3, 8.65,9.0 and 10.2.

Nnclear magnetic resonance spectrum peaks (PPM; CD01 1.25(s), 9H;1.45(d), 6H; 3.15(c), 2H; 4.44(s), 1H; 5.26-6.1(0), 5H; 7.0-7.66(c), 7H;and 7.7-9.2(c), 5H. 7

EXAMPLE 12 Employing the procedure of Example 11 and starting with theappropriate penicillin ester and requisite guanylureidoalkanoylchloride, the following compounds are prepared:

The procedure of Example 1 is again repeated, starting with theappropriate reagents, to provide the following penicillins of theD-configuration.

1 In this table, the Z group representing the bridging A group and theamino moiety is written in such a manner I that the left bond of thegroup is attached to the --(5 group one nitrogen removed from thepenicillin molecule 0 I and the right bond of the group to the 5 of theurea moiety.

6 [D-2-Phenyl-2- (3-guanyl-1-ureidoacetamido) acet- EXAMPLE 14amidoJpenicillanic acid sodium salt To a slurry of g. (0.061 mole) of 6-[D-2-phenyl-2- (3-guanyl-l-ureidoacetamido)acetamido1penicillanic acidin 100 ml. of dioxane and ml. of water (cold) is added 5.2 g. (0.061mole) of sodium bicarbonate dissolved in ml. of water. The partialsolution is allowed to stir for 15 min., some insolubles filtered, andthe filtrate freeze-dried. The resulting solid is slurried in 2 l. ofchloroform, filtered and dried in vacuo, 29.2 g. (93% yield) EXAMPLE l56-[D-Z-Phenyl-Z-(3-guanyl-l-ureidoacetamido) acetamido1penicillanic acidtriethylamine salt EXAMPLE 16 6-[D-2-Phenyl-2-(a-{3-guanyl 1ureido}-I3-hydroxy- 20 benzylpenicillin triethylamine salt is added tothe filtrate. After stirring for 3 hrs. at room temperature, the mixtureis filtered and the filtrate added dropwise to 1.2 l. ofdipropionamido)acetamido1penicillanic acid (Ar=; ethyl ether. The solidsare collected, suspended in 50 5 ml. of methylene chloride and treatedwith 1 ml. of tri- CH ([311; ethylamine. After one hr. of stirring, theproduct is collected and dried in vacuo, 1.18 g. (76% yield). Infraredspectrum peaks (microns; KBr); 3.10, 5.72, R3 and f 6.05, 7.18, 7.65 and8.10.

T0 15 of y Y Q W Q contalfllng 345 10 Nuclear magnetic resonancespectrum peaks; (PPM; mg. (3 mmoles) of Nhydroxysuccmrmrde and 620 mg.0); 1,45, H 5,43; 7 43, 5 and 3 4 4 4 (3 mmoles) ofdicyclohexylcarbodiimide is added 676 mg. (3 mmoles) ofa-(3-guanyl-1-ureido)-I9-hydroxypro- EXAMPLE 17 pionic acidhydrochloride, and the resulting reaction mix- The procedure of Example15 is repeated, starting ture allowed to stir at room temperature for 2hrs. The 15 with the appropriate chemical reagents, to provide themixture is filtered, and 1.35 g. (3 mmoles) of D-a-aminofollowingcompounds of the DL-configuration:

S OH: .Ar-CHCONH- 1 i NH 5 o O-N--CO2H R JIL N/ /Ra NHIJ-NHJN\ Ar A R1R4 Ra a CoHs- I H- H- H- C H5- cnm- I H- H- CH;- CH;-

CQHF- I H- CH; H- CH3- CHaCHOHCH CoHs- I H H- (CH2)II 0111011011011-C6Hb H- H-- H- H HSCHzCH- CaHa- H- -(CH2)a H-- HSOHzCH- CaHt- I H- 011H- i-C4Hr- HSOHzOH- Ca a- I H- H- czHr- CzHs HSCHzCH- 01H5- I 11 H- H-11- CSHa(CHz)2CH- 4-HO CIIH4' I H- CzHs- H- CzHIs- CHaS(CH2)zCH- 4-11001H;- I H- H- CH CH1- HOCHzCH- 4-H O CoH4- I H- H- H-- H- HO(CHz)zCH-441005114 I CH3 H- H- OH;-

CH3S(CH2)zCH- 4-110 06H! I H- H- H- H- OHaCHOHCH- 4-110 onn- I H- CH3-CH CH3- CHzCHOHCH- 4-110 CaHi J: H- CH: CHs- CHr- HSCHz H 4-HOCaH (OH;H- H- 2-OIHIS- I H- H- H- H- z-omns- I H- H- CH;- CH:-

2-C4H3s- I CH3- CH; CH,- CH;

HSCHzCH- 2-C4H3S- I CH3- CH3- CH3- 0113- CHaSCHzCH- 2-ciHas- I H- H- H-H- CH3S(CHZ)2CH 2-C H3 S I H- -(CHz) s-' HSCHz0H- 3-C4HaS- H- H- H-- H-I HOCHzCH- 21 22 TABLE-Continued 3-0 H s- H- H- o 4 a HOOHAJH- 3-O H S-H- H- 2)a 3 H s CHaCHOHOH- H CH 4 3 CHaCHOHCH- 93 3-0 H s- H- CH H- CH 4a nsomc'xna 3-C4H3S I H- CH3- CH3- CH3 HSCHICH- a-mms- I H- H- H H-CHaS(OH2)2CH 3-0 H s- H- H- o H CH 0 H 4 a 0H3SCH2C|H a 6 2*- o 50H;

EXAMPLE 18 R4 6- [D2-Phenyl-2-(p-{3-guanyl-1-ureido}phenylacet- 2 0 1 mmo ll amido)ace 1d ]pen1c1llan1c acid To a solution of 4.5 g. (0.01 mole)of D-a-aminobenzylpenicillin triethylamine salt in 50 ml .of drydimethylformamide at ice bath temperatures is added 1.75 R2 R3 ml.(0.0125 mole) of triethylamine followed by p-(3- cm- H- H H-guanyl-1-ureido)phenylacetyl chloride hydrochloride (3.6 8 g: g: 8%: g.,0.0125 mole). The resulting suspension is allowed to n-C3H1 H- I-I CH3-stir for 1.5 hrs. at 10 C., and is subsequently filtered, 2 525 3 gf andthe filtrate added dropwise into a cold solution of H- (CH2)4 1.4 l. ofchloroform and 400 ml. of acetone. The resultgil E EJHQh ing precipitateis filtered and dried in vacuo, 5.78 g.

The infrared and nuclear magnetic spectra are consistent with thestructure of the isolated product. PREPARATION B A 3-Guanyl-1-nitr0ureascyanoguanidines l. 3-(N-Methylguanyl)-1-n1trourea 1. The previouslyknown cyanoguanidines of the for- 1-CYam-3-IPethY1gPamdme 0-056 mole)mula added slowly 111 portions to a mixture of 20 ml. of cone.

sulfuric acid and 8 m1. of cone. nitric acid at a temperature of 0-10 C.with continuous mechanical stirring. R When the addition is complete,the reaction mixture is stirred for 1 hr. at 0 C. and at 20 C. for 2hrs., and N finally added to 200 ml. of ice and water. The precipitate,N--N-CN which formed after several hours, is filtered, washed with R3 1acetone, and dried in vacuo, 4.2 g. (47% yield).

The above procedure, which is essentially the method as taught byFrankel, et al., J. Chem. Soc., 2698 (1967), is are prepared by thesynthetic procedure of the indicated employed to convert thecyanoguallldlnes of Preparation referencfi as follows: A-1 and to thefollowing, previously unreported, 3- 50 guanyl-l-mtroureas,intermediates for the sub ect compound of the present invention:

R1 R2 R3 R4 Reference R 4 H H- 0113- H- Kitawaki, Nippon Kagaku 0 on H Kli if 1 h H a on a mu H a a 272311; 85, 5? (1964)? N C N C NHNOJ H- CH3-CH CH3 Allenstein, Chem. Ber., 100, 2604 I H H OH H A i1 z l J on s R3R1 some a., em. 0c.,

0 11 H Bl h 1 599 713 R1 R4 R2 R3 H- o H- t s a H- 11 CZHZ- CgHyus. Pat.2,438,124. H- H 0H3- CHi H- H- i-C3H1 11- British Pat. 599,722. H- CH3CH1? s- H H- S'CQHQ H- British Pat.599,713. H- H- C2H5 H- H- CH3 GHQ-U.S. Pat. 2,438, 124. H- H- 02H5 C2H5 H- 12-0 11 woiH1- H-- British Pat.1,053,307. H- C2H5 CzH5 H H- i-CsHr- C2Hi-;- British Pat. 599,713. H- H-H- 1-C3H1- H- H- 0 3- CzH British Pat.599,713. H- H H-- s-C4 o H- H- E-t-O4H Gadekar, et al., J. Med. Chem. H- C a- H CH3 11,811 (1968). H- H-n-C3 7 71-0311 H (CH2)5 H- British Pat. 599,722. 02H? #02117- Hi-CiHri-CaH Birtwell, et 111., J. Chem. 800., H'- C2H5- H- CH3- 1645(1948). H- i-oiHr- H H- H- t-C4H CHa- Gadekar, at (11., J. Med. Chem, H--(GH2) 8110968) 1 Z'CSHI" t-CsH7- g5 [H3 t- C 4H9- OH:- H- H- CH3- o,H.-H- H OH3 2. The following cyanoguanidines, employed as interf g? 3Emediates leading to the products of the present invention 'n-C H H- i-OH and previously unknown in the chemical literature, are L 94 CH3 CH3 Cs CH3 synthesized by one or more of the procedures described CH3 H--(OH;).;

in the above references:

23 PREPARATION 11:1)

3-Guanyl-1 -14reid0alkan0ic Acids 1. 3-Guanyl-1-ureidoacetic acid Amixture of 24.0 g. (320 mmoles) of glycine and 46.8 g. (320 mmoles) ofB-guanyl-l-nitrourea in 960 ml. of water heated and stirred at 100 C.for 1.5 hrs. The reaction mixture, after standing at room temperatureovernight, is cooled in an ice bath, and the resulting precipitatedproduct filtered, washed successively with water, ethanol and ether, anddried in vacuo, 30.53 g., m.p. 203 C.

a. 3-guanyl-1-ureidoacetyl chloride hydrochloride.To 300 ml. of drymethylene chloride maintained under a nitrogen atmosphere is added 12.0g. (0.075 mole) of B-guanyl-l-ureidoacetic acid and 15.5 g. (0.075 mole)of phosphorous pentachloride and the mixture allowed to stir at roomtemperature overnight. The product is filtered, washed with drymethylene chloride and dried in vacuo, 14.0 g. The infrared spectrum isconsistent with the iso lated product.

b. 3-guanyl-l-ureidoacetic acid hydrochloride-A suspension of 1.36 g. of3-guanyl-1-ureidoacetic acid in 100 ml. of diethyl ether at roomtemperature is stirred while hydrogen chloride gas is slowly bubbledthrough the mixture. After fifteen minutes, the addition of gas isstopped and the resulting white powder filtered and dried in vacuo, 1.4g. Infrared spectrum of the product is consistent with completeconversion to the hydrochloride salt.

2. Following the procedure of Preparation C-l, which is essentially thatas taught by Frankel, et al., J. Chem. 800., 2698 (1967), and startingwith the requisite aminoacids and 3-guanyl-1-nitroureas fromPreparations B-1 and B-2, the following intermediates are prepared:

TABLEC0ntinued 3. 3-(N-Phenylguanyl)-1-ureidoacetic acid hydrochlorideTo 50 ml. of benzene containing 6.75 g. (0.05 mole) of N-phenylguanidineis added in portions 6.45 g. (0.05 mole) of ethyl isocyanatoacetate andthe resulting solution heated to reflux for 3 hrs. The reaction mixtureis cooled and the solvent removed in vacuo.

The residual intermediate, ethyl 3-(N-phenylguanyl)-l ureidoacetate istreated with 200 ml. of concentrated hydrochloric acid and the resultingsolution heated at steam bath temperature for 1-2 hrs. The mixture isconcentrated to dryness under reduced pressure, and the productrecrystallized from a small amount of water.

4. Starting with the appropriate guanidine and requisiteisocyanatoalkanoate, prepared from the corresponding aminoacid esterhydrochloride and phosgene according to the method of Humphlett, et al.,J. Org. Chem., 26, 2507 1961), and employing the procedure ofPreparation C-3, the following intermediates are synthesized:

5. 1- (N-Carboxymethylcarbamyl -2-amino-2-imidazoline hydrochloride a. 1(N-Ethoxycarbonylmethylcarbamyl)-2-mercapto- 2-imidazoline.A mixture of20.4 g. (0.2 mole) of 2- mercap'to-Z-imidazoline and 25.82 g. (0.2 mole)of ethyl isocyanatoacetate is heated to C. under nitrogen for 12 hrs.,at which time the mixture solidifies. The temperature is maintained foran additional 30 min., and the mixture is then cooled, triturated withacetone, filtered and dried in vacuo.

b. 1 (N-Carboxymethylcarbamyl)-2-mercapto-2-imidazoline.-A suspension of11.32 g. (0.049 mole) of the above ester in 29.6-ml. of 2N sodiumhydroxide solution is stirred at room temperature until the solids aredissolved (-2 hrs.). The solution is filtered and the filtrate acidifiedwith 6N hydrochloric acid to pH 2. The resulting precipitate isfiltered, washed with a small volume of water and dried in vacuo overphosphorous pentoxide.

c. 1-(N-Carboxymethylcarbamyl)-2-methylthio-2-imidazoline hydroiodide.Asuspension of 7.1 g. of the above acid and 3.2 ml. of methyl iodide in60 ml. of acetone is allowed to stir at room temperature overnightduring which time the starting materials gradually dissolve and theproduct precipitates. The mixture is poured into 150 ml. of ethyl etherand the solids are collected and dried in vacuo.

d. 1 (N-Carboxymethylcarbamyl)-2-amino-2-imidazoline.-An ethanol (22ml.) solution containing 3.04 g. (8.8 mmoles) of the aboveisothiouronium salt is treated with ammonia gas introduced below thesurface of the solution employing a gas dispersion tube. After 15-20min. the addition is stopped and the reaction mixture allowed to stir atroom temperature overnight. The resulting precipitate is filtered,washed with ethanol, then ether and dried in vacuo.

6. Starting with the requisite thiourea derivative andisocyanatoalkanoate, and repeating the general sequence of reaction inPreparation C-5a through C-Sd, the following intermediateguanylureidoalkanoic acid congeners are synthesized:

7. l-N-Benzylguanyl)-3-ureidoacetic acid.

a. 1 (N-Ethoxycarbonylmethylcarbamyl)thiourea-A mixture of 26.6 g.(0.205 mole) of ethyl isocyanatoacetate and 15.8 g. (0.2 mole) ofthiourea is heated to 100 C. for 35 min. under a nitrogen atmosphere,followed by cooling and trituration of the solids with acetone. Theproduct is filtered and dried in vacuo, 13.9 g.

b. 1-(N-Carboxymethylcarbamyl)thiourea-A suspension of 10.12 g. (.05mole) of the above ester in 30 ml. of 2N sodium hydroxide solution isstirred at room temperature until most of the solids are dissolved. Themixture is filtered and the filtrate acidified to pH 2.0. The resultingprecipitate is filtered, washed with a small amount of water and driedin vacuo over phosphorous pentoxide, 6.5 g.

c. 1 N-(Carboxymethylcarbamyl)-S-methylisothi0urea hydroiodide.-Anacetone (60 ml.) solution of 6.37 g. of the above acid is treated with3.2 ml. of methyl iodide and the solids, which gradually dissolved, areallowed to stir at room temperature overnight. The suspension is pouredinto 150 ml. of ethyl ether, and the product is filtered and dried, 11.0g.

d. 1 (N-Benzylguanyl)-3-ureido acetic acid.Two and eight-tenths grams(8.8 mmoles) of the S-methylthiourea above is dissolved in 25 ml. ofethanol and treated with 3.18 ml. (29 mmoles) of benzylamine. Afterseveral hours at room temperature a white percipitate commences to form.After stirring overnight at room temperature the product is filtered,washed with ethanol, then ether and dried in vacuo, 2.0 g.

8. Starting with the appropriately substituted thiourea andisocyanatoalkanoate, and repeating the procedure of Preparation C7athrough C-7d, the following intermediates are prepared:

9. 1-(2-Imidazolinyl)-3-carboxymethylurea a.l-(Ethoxycarbonylmethyl)-3-cyanourea.To a partial suspension of sodiumcyanamide formed from 4.2 g. (0.1 mole) of cyanamide and an equivalentamount of sodium hydride in 35 ml. of dry dimethylformamide is added12.9 g. (0.1 mole) of ethyl isocyanatoacetate dropwise. The reaction ismaintained at room temperature for several hours, after which themixture is poured into water, the pH adjusted to 5, and the productextracted with methylene chloride. The solvent layer is dried oversodium sulfate and concentrated to dryness, the product being employedin the next reaction without further purification.

b. l-(Ethoxycarbonylmethyl)-3-thiocarbamylurea.-A solution of 12.8 g.(0.075 mole) of the above ester in 50 ml. of ethanol is saturated withhydrogen sulfide gas and the solution heated to 50-60 C. for 3.5 hrs.The solvent is removed under reduced pressure and the residual productcrystallized from methanol.

c. 1 (Carboxymethyl) 3-thi0carbamylurea.-Twenty and five-tenths grams(0.1 mole) of the above ester is added to 60 ml. of 2N sodium hydroxidesolution and the mixture allowed to stir at room temperature until mostof the material has dissolved. The suspension is filtered and thefiltrate acidified to pH 2. The resulting product is filtered, washedwith 10 ml. of water and dried in vacuo.

d. 1-(N-Carboxymethylcarbamyl)-S-methylisothiourea hydroiodide.tAcetone(60 ml.) is charged with 8.9 g. (0.05 mole) of the above acid followedby the addition of 3.8 ml. of methyl ioide and the mixture allowed tostir at room temperature overnight. The precipitated product isfiltered, washed several times with ether and air dried.

e. 1 (Z-Imidazolinyl) 3 (carboxymethyl)urea.-To 18.6 g. (0.1 mole) ofthe above isothiourea in ml. of ethanol is added 12.0 g. (0.2 mole) ofethylenediamine and the reaction mixture warmed to 50-60 C. for severalhours and then allowed to stir at room temperature overnight. Theproduct, which precipitates from solution is filtered and recrystallizedseveral times from methanol.

10. Employing the procedure of Preparation C-9a through C-9e andstarting with the appropritae reagents, the followingguanylureidoalkanoic acids are synthesized:

28 ment with hydrogen chloride gas, 311 mg. of the desired product.

3. Starting with the appropriate dior tripeptide and requisite3-guanyl-1-nitrourea, and employing the procedure of Preparation D-1 and2, the following intermediates are prepared:

PREPARATION D 3-Guanyl-l-ureidoalkanoic acids (n=2 or 3) 1.3-Guanyl-l-ureidoacetylglycine hydrochloride A mixture of 1.32 g.mmoles) of glycylglycine and 1.47 g. (10 mmoles) of 3-guanyl-1-nitroureain ml. of water is heated to reflux overnight. The reaction mixture iscooled, and the solids filtered and dried in vacuo, 875 mg. The infraredspectrum of the product is consistent with the assigned structure.

To a suspension of the above compound in 5 ml. of diethyl ether is addedvia a gas dispersion tube dry hydrogen chloride gas. After min. the gasaddition is stopped and the precipitate allowed to stir for 3 hrs. Theproduct is filtered under nitrogen and dried in vacuo.

2. 3-Guany1-l-ureidoacetylglycylglycine hydrochloride In a mannersimilar to Preparation D-l, 1.87 g. (0.01 mole) of glycylglycylglycineand 1.47 g. (0.01 mole) of S-guanyl-l-nitrourea in 15 ml. of wateryields, on treat- 4. [i- (,8-[3-{N-Benzyl-N-phenylguanyl}-l-ureido]propionoyl alanine To a partial suspension of 1.9 g. (0.01 mole) offl-alanine triethylamine salt in 25 ml. of dry dimethylformamide at roomtemperature, under a nitrogen atmosphere and cooled to 0 C. in an icebath is added 1 g. (0.01 mole) of triethylamine followed by theportionwise addition of 3.95 g. (0.01 mole) of 3-(N-benzyl-'N-phenylguanyl)-1-ureid0propionyl chloride hydrochloride. The mixture isstirred vigorously for 1-2 hrs., and is then allowed to warm to roomtemperature. The mixture is filtered, and the filtrate added to ml. ofdiethyl ether. The resulting precipitate is filtered, reslurried in asmall volume of water, refiltered and dried in mom 5. Employing thegeneral procedure of Preparation D-4 and starting with the appropriateguanylureidoalkanoyl chloride and amino acid or dipeptide, the followingintermediates are prepared:

R and R when considered together are alkylene containing from 2 to 4carbon atoms; and

I'M if i /NCIN-C[NH-AC ]..0H R: R:

A '01 R1 R4 R2 R3 -(CH2)4-- 2 H- H- CsHsCHz- CH2- 2)r 3 (CH2)l---(CH2)5- cum- 2 H (GHz) 11- CHQCHaH- 2 H- H- C|HCH3- CQHsCHr- CHaC IHF-3 H- (CH1): 11-

(CH2):- 3 C HsCHr- H- H- H- CH:(CH2)2(3H 2 ---(CH2): 3- CH;-

(CH2)2CH(CH3)- 2 H- H- CQH; CuHs- --(oH2)1- 3 H- c.H,oH2- H- 0.115

CH3(CH2):(5H- 2 H- H- C2H5- C Hs -(CH2)3' 2 2)e- PREPARATION E R and Rwhen considered together are alkylene containing from 4 to 5 carbonatoms. Am m0 Acfds and DI i Trlpepndes 2. A compound of claim 1 havingthe D configuration. 1. The am no acids and peptides employed as t 3. Acompound of claim 2 wherein Ar is phenyl, n is 1, medites leadlng t0 theProducts of the Present mvelltl on A is selected from the groupconsisting of alkylene conare either commercial reagents or are preparedaccording mi i fro 1 to 4 carbon atoms and alkylidene i to literatureprocedures well known to those skilled in the ing from 2 to 4 carbonatoms, and R R and R are each art, for example, according to thesynthetic routes as hydrogen, taught y Greenstein and Winitz in m y Ofthe 4. The compound of claim 3 whereinAis CH and Amino Acids, John Wiley& Sons, Inc., New York, N.Y., R is CH 1961, Vols. 1, 2 and 3. 5. Thecompound of claim 3' wherein A is -CH,, and

What is claimed is: R is hydrogen. 1. A compound selected from those ofthe formula: 6. The compound of claim 3 wherein A is -CH CH;;

S CHa and R is hydrogen. A -CHCQNH 7. A compound of claim 2 wherein n is1, A is alkylene 1 containing from 1 to 4 carbon atoms and R R R and Q-1 021.1 R are each hydrogen. 8- The compound of claim 7 wherein Ar is2-thienyl l andAisCH 1 34 9. A compound of claim 2 wherein AI is phenyl,A is R alkylene containing from 1 to 4 carbon atoms and R R 1 R and Rare each hydrogen. 10. The compound of claim 9 wherein n is 2 and A is 1'I h dfl'9h' '3dA' and the pharmaceutically acceptable salts thereof,wherein e compoun o 6 mm w emu an 18 Ar is selected from the groupconsisting of phenyl, 4-hy- References Cited droxyphenyl, 2-thienyl and3-thienyl; A is selected from the group consisting of alkylene con-UNITED STATES PATENTS taining from 1 to 4 carbon atoms and alkylidenecon- 3,579,501 5 71 McGregor 0 239 1 f p from 2 to 4 carbon atoms;3,483,188 12/1969 McGregor 260239.1 n is an integer of 1 to 3; R R R andR when considered separately are each N s RIZZO, P i ary Examinerselected from the group consisting of hydrogen and alkyl containing from1 to 4 carbon atoms; US. Cl. X.R. R and R when considered together arealkylene contain 424-271 ing from 2 to 4 carbon atoms;

